Human embryonic stem cells (hESCs) offer a potentially unlimited source of tissue for replacement therapy in type 1 diabetes. However, current differentiation protocols like the 5-stage Novocell protocol yield few insulin-producing cells, which in vitro are unresponsive to glucose. Only after in vivo transplantation do the cells acquire glucose responsiveness. The molecular switches that turn on glucose responsiveness remain a mystery. Recent studies highlight a central role of small noncoding RNAs called microRNA (miRNA) in the regulation of gene expression during development. The overarching goals of this proposal are to develop a rapid screen for stem cell- derived insulin-positive cells that respond to glucose and to characterize the genetic and epigenetic (miRNA-based) mechanisms important for acquisition of this important phenotype. As a starting point for this proposal, we have already conducted a miRNA microarray analysis on populations of stage-5-differentiated hESCs vs. human islet cells. Although both populations are known to be composed of diverse cell types, our preliminary analysis showed striking differences in the level of several miRNAs, including miR-375 - a miRNA previously shown by other groups to be important for maintaining pancreatic beta cell mass, and, thus, lending support to the idea that we may find miRNAs that regulate beta-cell-specific genes, including those regulating glucose responsiveness. We have also initiated development of a novel fluorescent reporter that identifies insulin-producing cells, as well as reports insulin secretion. Here we outline a systematic approach to test the role of miRNAs in regulating glucose responsiveness. Our specific aims are: 1. Develop a fluorescent reporter that both identifies insulin-positive cells and reports insulin secretion, as a tool to rapidly screen new protocols to activate glucose-response genes. 2. Identify miRNA candidates for regulation of glucose-response genes. We will express the fluorescent reporter in Stage-4 or -5 hESCs and use fluorescence-activated cell sorting (FACS) to isolate insulin-positive but glucose non-responsive Stage-5 hESC-derived cells. We will compare their miRNA profiles with human beta cells using a differential miRNA microarray approach. 3. Test the functional activity of candidate regulatory miRNAs using population and single-cell physiological and genetic analysis. PUBLIC HEALTH RELEVANCE: Type 1 diabetes treatment by human embryonic stem cells has been blocked so far, because, while scientists can coax the stem cells to turn into cells that make insulin, those cells don<t release insulin when exposed to sugar. The aim of this work is to mark the insulin-making cells with a fluorescent marker that can tell us quickly whether a cell is secreting when exposed to sugar. This marker will help us to work out ways to turn on the cell<s response to sugars, and it will help us to prove that insulin is being secreted appropriately in response to sugar stimulation.